E. coli NusA protein binds in vitro to an RNA sequence immediately upstream of the boxA signal of bacteriophage lambda.

Tsugawa, Akiko; Kurihara, Tatsuya; Zuber, Mohammed; Court, Donald L.; Nakamura, Yoshikazu

doi:10.1002/j.1460-2075.1985.tb03935.x

Cited by 31 publications

(15 citation statements)

References 18 publications

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“…In agreement with previous work (14), N (Fig. 4A, lanes c and e), somewhat contrary to reports that NusA binds to N (29) and also RNA (30 RJAPN mA~~~~2 70 nLt RNAP+NusA ,,,,,,,,,,,,,,,,,,7rl RNAP+riusA+N lllllllll4|||0,,,,,,,, demonstrating the basal, NusA-independent antitermination by N (16). Together, they indicate that NusA and boxB RNA cooperate and facilitate a direct, productive interaction between N and RNAP.…”

Section: Methodssupporting

confidence: 90%

Bipartite function of a small RNA hairpin in transcription antitermination in bacteriophage lambda.

Chattopadhyay

García‐Mena

DeVito

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

104

119

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Transcription of downstream genes in the early operons of phage A requires a promoter-proximal element known as nut. This site acts in cis in the form of RNA to assemble a transcription antitermination complex which is composed of A N protein and at least four host factors. The nut-site RNA contains a small stem-loop structure called boxB. Here, we show that boxB RNA binds to N protein with high affinity and specificity. While N binding is confined to the 5' subdomain of the stem-loop, specific N recognition relies on both an intact stem-loop structure and two critical nucleotides in the pentamer loop. Substitutions of these nucleotides affect both N binding and antitermination. Remarkably, substitutions of other loop nucleotides also diminish antitermination in vivo, yet they have no detectable effect on N binding in vitro. These 3' loop mutants fail to support antitermination in a minimal system with RNA polymerase (RNAP), N, and the host factor NusA. Furthermore, the ability of NusA to stimulate the formation of the RNAP-boxB-N complex is diminished with these mutants. Hence, we suggest that boxB RNA performs two critical functions in antitermination. First, boxB binds to N and secures it near RNAP to enhance their interaction, presumably by increasing the local concentration of N. Second, boxB cooperates with NusA, most likely to bring N and RNAP in close contact and transform RNAP to the termination-resistant state.The positive control of genes that facilitate the bimodal development of A and related phages in Escherichia coli depends on two distinct operon-specific antiterminators (1). The N antiterminator activates the early operons, whereas the Q antiterminator activates the late operon. Both proteins function by a common mechanism: they capture RNA polymerase (RNAP) during early phases of transcription and mask RNAP's response to the downstream terminators (2-8). However, each antiterminator recognizes the respective genetic signal and captures RNAP by distinct mechanisms. The signals for Q action span the late promoter and the early transcribed region. Q binds to a DNA sequence within the late promoter and acts upon RNAP paused at a defined site (9). Specific nucleotides in the nontemplate strand of this region interact with RNAP not only to induce pausing but also to endow upon RNAP the conformation that is essential for engagement by Q (10). In contrast, the nut site, required for N action, functions in the form of . It can facilitate the productive interaction between N and RNAP at remote sites, suggesting that nut RNA may act similarly to DNA enhancers, binding N and delivering N to RNAP through RNA looping (11). Finally, while a single host factor (NusA) appears to be sufficient for Q activity, processive antitermination by N demands three additional factors: NusB, S10 ribosomal protein (NusE), and NusG (2, 14-16).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §173...

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Section: Methodssupporting

confidence: 90%

Bipartite function of a small RNA hairpin in transcription antitermination in bacteriophage lambda.

Chattopadhyay

García‐Mena

DeVito

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

104

119

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“…NusA also interacts with K N protein, RNA, E. coli rho factor, and perhaps nusB protein (10,15,18,47,50,52). The pleiotropic roles of NusA during transcription may be attributed to the capacity for multiple interactions with these regulatory components.…”

mentioning

confidence: 99%

Genetic interaction between the beta' subunit of RNA polymerase and the arginine-rich domain of Escherichia coli nusA protein

1991

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The nusAl) mutation causes reduced transcription termination and temperature-sensitive growth of Escherichia coil. Suppressor mutations that restored growth of nusAll mutant cells were isolated and named sna mutations. The intergenic suppressor mutation sna-10 was located in the rpoC gene at 90 min, which encodes the (3' subunit of RNA polymerase. sna-10 complemented the defect in tRl termination caused by nusAl) and by itself stimulated termination of transcription at the X tRl terminator. sna-10 is specific to the nusAl) allele and unable to suppress cold-sensitive growth of the nusAlO mutant. nusA10 carried two base substitutions at positions 311 and 634, causing two amino acid changes from the wild-type sequence. During these studies, we found three -1 frameshift errors in the wild-type nusA sequence; the correct sequence was confirmed by the peptide sequence and gene fusion analyses. The revised sequence revealed that nusAl and nusAl) are located in an arginine-rich peptide region and substitute arginine and aspartate for leucine 183 and glycine 181, respectively. The intragenic suppressor study indicated that the nusAl) mutation can be suppressed by cha ng the mutated aspartate 181 to alanine or changing aspartate 84 to tyrosine.

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“…If boxA-like sequences serve as recognition sites for NusA (13,18,29,30,38), then this implies that NusA acts as a transcription termination factor at tnaL (37). In its simplest form, this hypothesis predicts that templates carrying tnaC alterations should produce wild-type transcription pausing and termination patterns in the absence of NusA, but should show altered patterns in the presence of NusA.…”

mentioning

confidence: 99%

Rho-dependent transcription termination in the tryptophanase operon leader region of Escherichia coli K-12

Stewart¹,

Landick²,

Yanofsky³

1986

J Bacteriol

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Recent studies have suggested that expression of the tryptophanase (tna) operon of Escherichia coli is subject to transcription termination-antitermination control (V. Stewart and C. Yanofsky, J. Bacteriol. 164:731-740, 1985). In vivo studies have indicated that the transcribed leader region, tnaL, contains a site or sites of rho-dependent transcription termination (rho is the polypeptide product of the gene rho). We now report direct in vitro evidence that tnaL contains rho-dependent termination sites. In vivo termination appeared to occur at the rho-dependent termination sites identified in vitro. Transcription pausing analyses correlated sites of pausing in tnaL with sites of rho-dependent termination.

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E. coli NusA protein binds in vitro to an RNA sequence immediately upstream of the boxA signal of bacteriophage lambda.

Cited by 31 publications

References 18 publications

Bipartite function of a small RNA hairpin in transcription antitermination in bacteriophage lambda.

Bipartite function of a small RNA hairpin in transcription antitermination in bacteriophage lambda.

Genetic interaction between the beta' subunit of RNA polymerase and the arginine-rich domain of Escherichia coli nusA protein

Rho-dependent transcription termination in the tryptophanase operon leader region of Escherichia coli K-12

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